The functional RNA domain 5BSL3.2 within the NS5B coding sequence influences hepatitis C virus IRES-mediated translation

Romero-López, Cristina; Berzal‐Herranz, Alfredo

doi:10.1007/s00018-011-0729-z

Cited by 58 publications

(119 citation statements)

References 60 publications

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“…In modified translation only reporter systems an antisense-LNA, complementary to both the terminal and bulge loop of SL9266, inhibited HCV IRES driven translation ∼2-fold more efficiently than for poliovirus IRES initiated translation and ∼4-fold more efficiently than m 7 G cap-dependent translation. The sub-terminal bulge loop of SL9266 and domain IIId of the HCV IRES include a six-nucleotide complementary motif (40). Their demonstrated interaction in in vitro assays has been suggested to indicate a role in modulation of translation (28,40), although we have not found evidence to support this model or interaction using SHAPE mapping and reverse genetics (23).…”

Section: Discussionmentioning

confidence: 99%

“…The sub-terminal bulge loop of SL9266 and domain IIId of the HCV IRES include a six-nucleotide complementary motif (40). Their demonstrated interaction in in vitro assays has been suggested to indicate a role in modulation of translation (28,40), although we have not found evidence to support this model or interaction using SHAPE mapping and reverse genetics (23). The fact that blocking SL9266/PK significantly inhibits translation from both a m 7 G cap and poliovirus IRES - although to a lesser degree than from the HCV IRES - further suggests that SL9266/PK does not act on translation through a direct RNA–RNA interaction with the HCV IRES (the poliovirus IRES does not contain sequence motifs complementary to known interactions of the bulge-loop of SL9266 using either canonical or non-canonical pairing; data not shown).…”

Section: Discussionmentioning

confidence: 99%

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Inhibition of HCV translation by disrupting the structure and interactions of the viral CRE and 3′ X-tail

Tuplin

Struthers

Cook

et al. 2015

Nucleic Acids Research

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A phylogenetically conserved RNA structure within the NS5B coding region of hepatitis C virus functions as a cis-replicating element (CRE). Integrity of this CRE, designated SL9266 (alternatively 5BSL3.2), is critical for genome replication. SL9266 forms the core of an extended pseudoknot, designated SL9266/PK, involving long distance RNA–RNA interactions between unpaired loops of SL9266 and distal regions of the genome. Previous studies demonstrated that SL9266/PK is dynamic, with ‘open’ and ‘closed’ conformations predicted to have distinct functions during virus replication. Using a combination of site-directed mutagenesis and locked nucleic acids (LNA) complementary to defined domains of SL9266 and its interacting regions, we have explored the influence of this structure on genome translation and replication. We demonstrate that LNAs which block formation of the closed conformation inhibit genome translation. Inhibition was at least partly independent of the initiation mechanism, whether driven by homologous or heterologous internal ribosome entry sites or from a capped message. Provision of SL9266/PK in trans relieved translational inhibition, and mutational analysis implied a mechanism in which the closed conformation recruits a cellular factor that would otherwise suppresses translation. We propose that SL9266/PK functions as a temporal switch, modulating the mutually incompatible processes of translation and replication.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Inhibition of HCV translation by disrupting the structure and interactions of the viral CRE and 3′ X-tail

Tuplin

Struthers

Cook

et al. 2015

Nucleic Acids Research

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“…The stem-loop 5BSL3.2 within the CRE region forms a high-order structure involved in viral RNA translation and replication (31). Six nucleotides form base pairs between the bulge of the 5BSL3.2 stem-loop and the IIId loop of the IRES, inhibiting translation initiation (32). Conversely, mutations in 5BSL3.2 increased IRES activity supporting the existence of a functional high-order structure in the HCV genome that involves two conserved RNA motifs, one within the IRES and another in the CRE region.…”

Section: Introductionmentioning

confidence: 99%

In-cell SHAPE uncovers dynamic interactions between the untranslated regions of the foot-and-mouth disease virus RNA

2016

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The genome of RNA viruses folds into 3D structures that include long-range RNA–RNA interactions relevant to control critical steps of the viral cycle. In particular, initiation of translation driven by the IRES element of foot-and-mouth disease virus is stimulated by the 3΄UTR. Here we sought to investigate the RNA local flexibility of the IRES element and the 3΄UTR in living cells. The SHAPE reactivity observed in vivo showed statistically significant differences compared to the free RNA, revealing protected or exposed positions within the IRES and the 3΄UTR. Importantly, the IRES local flexibility was modified in the presence of the 3΄UTR, showing significant protections at residues upstream from the functional start codon. Conversely, presence of the IRES element in cis altered the 3΄UTR local flexibility leading to an overall enhanced reactivity. Unlike the reactivity changes observed in the IRES element, the SHAPE differences of the 3΄UTR were large but not statistically significant, suggesting multiple dynamic RNA interactions. These results were supported by covariation analysis, which predicted IRES-3΄UTR conserved helices in agreement with the protections observed by SHAPE probing. Mutational analysis suggested that disruption of one of these interactions could be compensated by alternative base pairings, providing direct evidences for dynamic long-range interactions between these distant elements of the viral genome.

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“…These long-range interactions formed by stem-loop structures have been shown to be critical for HCV genome replication. It has been recently reported that the 5BSL3.2 CRE is involved in a long-range interaction with subdomain IIId of the 5= NTR IRES region, and the resulting interaction negatively influences the IRES-mediated translation (61,62). It is postulated that the 5BSL3.2 CRE could function as a switch modulating the translation and genome replication of the sense-stranded HCV genome (60).…”

mentioning

confidence: 99%

Systematic Analysis of Enhancer and Critical cis -Acting RNA Elements in the Protein-Encoding Region of the Hepatitis C Virus Genome

Chu

Ren

et al. 2013

J Virol

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g Hepatitis C virus (HCV) causes chronic hepatitis, cirrhosis, and liver cancer. cis-acting RNA elements of the HCV genome are critical for translation initiation and replication of the viral genome. We hypothesized that the coding regions of nonstructural proteins harbor enhancer and essential cis-acting replication elements (CRE). In order to experimentally identify new cis RNA elements, we utilized an unbiased approach to introduce synonymous substitutions. The HCV genome coding for nonstructural proteins (nucleotide positions 3872 to 9097) was divided into 17 contiguous segments. The wobble nucleotide positions of each codon were replaced, resulting in 33% to 41% nucleotide changes. The HCV genome containing one of each of 17 mutant segments (S1 to S17) was tested for genome replication and infectivity. We observed that silent mutations in segment 13 (S13) (nucleotides [nt] 7457 to 7786), S14 (nt 7787 to 8113), S15 (nt 8114 to 8440), S16 (nt 8441 to 8767), and S17 (nt 8768 to 9097) resulted in impaired genome replication, suggesting CRE structures are enriched in the NS5B region. Subsequent high-resolution mutational analysis of NS5B (nt 7787 to 9289) using approximately 51-nucleotide contiguous subsegment mutant viruses having synonymous mutations revealed that subsegments SS8195-8245, SS8654-8704, and SS9011-9061 were required for efficient viral growth, suggesting that these regions act as enhancer elements. Covariant nucleotide substitution analysis of a stem-loop, JFH-SL9098, revealed the formation of an extended stem structure, which we designated JFH-SL9074. We have identified new enhancer RNA elements and an extended stem-loop in the NS5B coding region. Genetic modification of enhancer RNA elements can be utilized for designing attenuated HCV vaccine candidates.

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The functional RNA domain 5BSL3.2 within the NS5B coding sequence influences hepatitis C virus IRES-mediated translation

Cited by 58 publications

References 60 publications

Inhibition of HCV translation by disrupting the structure and interactions of the viral CRE and 3′ X-tail

Inhibition of HCV translation by disrupting the structure and interactions of the viral CRE and 3′ X-tail

In-cell SHAPE uncovers dynamic interactions between the untranslated regions of the foot-and-mouth disease virus RNA

Systematic Analysis of Enhancer and Critical cis -Acting RNA Elements in the Protein-Encoding Region of the Hepatitis C Virus Genome

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